Commercial production of various medically and industrially valuable recombinant proteins by microbes is one the key challenges of modem biotechnology. Even though such systems are known, there are severe technical problems which are encountered within large-scale exploitation of microbial cell machinery. There are several secretion systems in Gram-negative bacteria which can be potentially exploited for secretion of recombinant heterologous proteins. The systems are briefly reviewed below.
1. Secretion Across the Inner Membrane
The majority of secreted proteins in Escherichia coli are synthesized as precursors with a classic N-terminal signal peptide (SP) which is essential for efficient export and which is cleaved during or following translocation across the inner membrane. Translocation is mediated by the Sec translocase (SecA/Y/G/E). SecA is a peripherally associated ATPase, which interacts with the signal sequence and mature part of the precursor, guides the polypeptide into the translocator and provides energy for the process. Sec Y, E and G are integral membrane proteins that form the translocator itself and a central aqueous channel through which the polypeptide is translocated. SecD and F have large periplasmic domains. Proposed functions of these two proteins are related to later steps in the process and have included release from the membrane and mediation of transfer of energy from the proton motive force. Polypeptides are translocated in an ‘unfolded’ state. Hence SecB is a cytosolic chaperone apparently dedicated to the secretion pathway which is required for export of a subset of secreted proteins. SecB both inhibits premature folding and targets the precursor to the membrane translocase complex. It now appears that basic principles of the export system are universal. Although translocation is primarily co-translational in eukaryotic systems and targeting to the secretion apparatus is primarily via SRP (signal recognition particle), the core translocator is homologous in both systems (yeast Sec 61 α and γ are homologous of E. coli SecY/E). Also, E. coli ffh and 4.5S RNA are homologous of eukaryotic 54 KD subunit and 7S RNA of SRP, respectively. Comparison of the eukaryotic and prokaryotic systems has been extensively reviewed recently (Rapoport, T., et al. (1996)Annual Review of Biochemistry. 65:271-303; Schatz, G., and B. Dobberstein (1996)Science. 271:1519-1526).
Similarities in the basic function of eukaryotic and bacterial export systems have meant that some mammalian proteins can be successfully secreted to the periplasm of E. coli. Examples include human insulin. Often, however, fine tuning is required such as optimising the N-terminus of the mature protein, removal of positively charged residues at the end of the SP or beginning of the mature protein, ensuring presence of a good cleavage site. Frequently, a bacterial signal peptide such as the OmpA SP has been used. The Caf1 signal sequence has also been successfully used to export mammalian cytokines (see below). A major problem on recombinant expression in E. coli is incorrect folding with accompanying protein degradation or accumulation in an insoluble and inactive form as inclusion bodies.
In addition to the sec dependent secretion system there are at least two other systems of protein translocation across the bacterial inner membrane. The M13 phage coat protein is also synthesised with an additional SP, but assembly of this protein across the membrane is independent of the sec machinery. Recently, a novel pathway involved in secretion of cofactor-containing proteins has been elucidated (Santini, G., et al. (1998) EMBO Journal, 17:101-112; Weiner, J., et al. (1998) Cell. 93:93-101). Proteins following this pathway have a long leader containing a characteristic ‘twin arginine’ motif. It is proposed that cofactor attachment occurs in the cytosol and that the fully folded protein is translocated across the inner membrane via products of the mttABC operon. In addition, there are cytosolic proteins of E. coli which appear to be localised in a priviledged site which is sensitive to osmotic shock. Therefore may have some transient access to the periplasm. Such proteins include thioredoxin (Lunn, C. A., and V. P. Pigiet. (1982) J.Biol.Chem. 257:11424-11430) involved in disulphide reduction of cellular components, the cytosolic chaperone DnaK (Yaagoubi, A.,et al. (1994) Journal of Bacteriology. 176:7074-7078), elongation factor Tu (Jacobson, G., et al. (1976) Biochemistry. 15:2297-2303) and inner membrane bound components of enterobactin synthase complex (Hantash, F., et al. (1997) Microbiology. 143:147-156), and capsule assembly (Rigg, G., et al. (1998) Microbiology. 144:2905-2914).
It has been suggested that this ‘compartment’ may be related to transient formation of adhesion zones between the bacterial inner and outer membranes, but nothing is known regarding properties of the protein which targets them to this location nor about the physical nature of this ‘compartment’. A number of cytosolic recombinant proteins (i.e. without SP) also behave in a similar manner and are thus presumably targeted to the same cellular location. These include GST fusion proteins, interleukin 1β (Joseph-Liauzun, E., et al. (1990) Gene. 86:291-295).
2. Extracellular Secretion Systems
Six different pathways for export of extracellular proteins have been identified in Gram negative bacteria. Each pathway has been identified in a diverse range of bacteria. The basic properties of these systems are summarised in FIG. 1 (recently reviewed by Lory (Lory, S. (1998) Current Opinion in Microbiology. 1:27-35).
The Type II pathway, which is considered to be the main terminal branch of the sec- dependent pathway, is used for export of many different unrelated soluble proteins. It involves a folded periplasmic intermediate and requires approximately 12 dedicated genes for export across the OM. Alternative terminal branches to the sec pathway include specific chaperone-dependent fimbriae assembly and the OM helper pathway. The former pathway also involves a periplasmic intermediate (at least partially folded) but in this case the secreted polypeptide is specifically transported in association with its own chaperone/outer membrane usher protein system. Outer membrane helpers fold into the outer membrane with concomitant exposure of the effector domain at the cell surface and, in the case of IgA protease, release via self-hydrolysis. Interaction with general periplasmic chaperones e.g. DsbA has been demonstrated as a critical step in secretion pathway for a number of sec dependent proteins.
Type I, Type III, and most members of Type IV pathways are sec independent and mediate secretion of a specific protein (subset of proteins or DNA (Type IV) directly from the cytosol. Type I results in secretion into the external media, whereas Type III targets the secreted protein directly into the eukaryotic cell following contact-stimulated activation of the secretion system. The Type III pathway also shares many features with flagellar assembly systems.
3. Secretion of Recombinant Heterologous Proteins in Gram-negative Bacteria
Incorrect folding of proteins in the cytosol may lead to degradation or formation of misfolded protein as inclusion bodies. In many instances, therefore, it is desirable to have heterologous expression of recombinant proteins in the bacterial periplasm, at the cell surface, or in the extracellular media, permitting correct folding and formation of a functional product. Proteins secreted to the periplasm of E. coli are in an oxidising environment, compared to the reducing environment of the cytosol. The periplasm contains oxidoreductases and chaperones (disulphide bond isomerase, DsbA and C, peptidyl prolyl cis-transisomerase, RotA. SurA, and FkpA) which are essential for the correct folding of proteins (Missiakas. D., and S.Raina. (1997) Journal of Bacteriology 179:2465-2471). In addition, recombinant proteins expressed in the periplasm or secreted to the extracellular medium would represent a high percentage of the final protein content of these respective compartments. Thus, when the final goal is to obtain a purified recombinant product, secretion of the product to the periplasm or externally should greatly facilitate purification protocols. Although there are quite a few systems available for periplasmic localisation of proteins, there is no major system for secretion of extracellular products from E. coli. Over the past decade there has also been a great deal of interest in expressing proteins and peptides on the surface of microorganisms. Phage display technology (Winter, G., et al. (1994) Annual Review of Immunology 12:433-455) utilises the coat protein of filamentous bacteriophage for surface display of proteins or peptides. Such technology has been applied to the isolation of specific antibody fragments and for the rapid identification of peptide ligands. Interest in surface display in E. coli (Georgiou, G., et al. (1993) Trends in Biotechnology. 11:6-10) and other Gram negative bacteria has centered around identification of protective epitopes and their applications as live vaccines, production of bacterial adsorbents and whole-cell biocatalysts.
Although there has been some success in expressing of proteins, there are a number of limitations within the existing systems as outlined below.
Most secretory/assembly pathways of E. coli have been investigated for their potential exploitation as secretion vehicles for heterologous proteins. These include systems that direct the protein to the periplasm, cell surface or extracellular medium.
3.1 SP Alone
A number of expression vectors use a bacterial SP (often that of the OM protein OmpA) to mediate export across the inner membrane. Destination of the protein depends on the nature of the protein itself. It is not uncommon for proteins exported in this way in high levels to form insoluble complexes, inclusion bodies, in the periplasm as a result of incomplete folding.
3.2. Affinity Purification Systems
Fusion expression systems have been developed to facilitate downstream purification of recombinant products. Examples include insertion of a His tag for purification on a Nickel column (Clontech, Qiagen, In vitrogen); fusion to MalE (New England Biolabs), maltose binding protein, with subsequent purification on an amylose column; thioredoxin fusions with PAO (phenyl arsine oxide) resin and chitin binding domain fusions with chitin columns (New England Biolabs). By inclusion or omission of SP in the vector, some of these systems (e.g. MalE, His Tag) can be adapted for periplasmic or cytosolic expression, respectively. In general, such vectors contain a highly specific protease cleavage site for downstream purification of the product. Fusions functional in both domains, e.g. MalE and secreted domain, can be obtained. This, however, is dependent on the nature of the protein. The carrier domain may interfere with folding of the recombinant protein resulting in protein degradation, insolubility of the protein due to membrane association or formation of insoluble inclusion bodies at higher concentrations.
3.3. Surface Display in E. coli 
Insertion of epitopes into major OM proteins (OmpA, LamB, PhoE), flagella, fimbriae. These systems involve insertion of epitopes into a permissive site, i.e. surface loop within OM proteins or flagellar, fimbriae subunits, without affecting assembly of the membrane protein or surface appendage. In general, there are severe size restrictions of the insert (10-60 amino acids) to avoid effects on folding and assembly of the protein. There are reports of surface display of whole proteins by preparing terminal fusions to part of the outer membrane protein. OmpA or of IgA protease. Using a Lpp- OmpA vector, complete enzymes have been localised to the surface of E. coli offering the potential of surface display, but these constructs lead to disruption of the outer membrane with concomitant toxicity to the cell and leakage of periplasmic contents. In addition, the fusion proteins follow the outer membrane protein assembly pathway. This limits the maximum number of surface molecules and more importantly it is evident that completely folded proteins possessing disulphide bonds cannot be assembled across the outer membrane by this route (Klauser, T., et al. (1990) EMBO Journal. 9:1991-1999; Stathopoulos, C., et al. (1996) Applied Microbiology and Biotechnology. 45:112-119).
3.4. Extracellular Secretion.
There have been limited reports on extracellular secretion of unrelated proteins by some of the above mentioned secretion pathways. The Hly Type I secretion pathway has been adapted to delivery of heterologous antigens (Gentschev, I, et al. (1996) Gene 179:133-140). Although apparently successful, this system delivers proteins directly from the cytosol and would preclude any protein which require exposure to the periplasmic space for correct folding, e.g. disulphide bond formation.
It is summarised below some of the serious drawbacks associated with recombinant protein expression:
(i) Periplasmic expression systems: Many heterologous polypeptides expressed in E. coli are either degraded or form aggregates and inclusion bodies as a result of incorrect folding. This may occur despite targeting of the protein to a preferred location, i.e. the cytosol (with a more reducing environment) or the periplasm (with a more oxidising environment and specific chaperones involved in folding). Employment of a signal sequence to proteins targeted to the periplasm results in varying degrees of efficiency of precursor processing, completion of translocation and correct folding. Some incorrectly folded proteins remain associated with the inner membrane and induce toxicity. In addition, they are extensively degraded resulting in a poor yield. Others accumulate in a non-native conformation as insoluble aggregates. Systems employing fusion proteins are available. These may to some degree enhance solubility of some recombinant proteins but others remain insoluble due to incomplete folding of the heterologous domain. A system leading to stimulation of the early folding event following translocation across the inner membrane would clearly enable periplasmic expression of many heterologous polypeptides which have thus far eluded successful expression in E. coli. 
(ii) Surface localisation in Gram negative bacteria: Generally, there is a strict limitation in the size of epitopes which can be expressed at the cell surface using proven surface expression vectors. Systems that permit surface expression of whole domains or proteins by fusion them to an outer membrane protein lead to membrane permeabilisation, periplasmic leakage and toxicity. In addition, there are limitations on the extent to which proteins can be folded if they are to be exported by this pathway. Finally, as these systems all use integral membrane proteins, they are limited with respect to the maximum expression level and would be very laborious to purify.